1. Field of the Invention
The invention is directed assays of bacterial endospore levels.
2. Description of the Prior Art
The prior art for species-specific bacterial spore detection, using the lateral flow immunoassay method, is based on observing the red color of gold nanoparticles. It uses two antibodies, in combination, to specifically detect the bacterial spore species of interest in solution. One of the antibodies is attached to a colloidal gold nanoparticle, and the other antibody is immobilized on the nitrocellulose membrane downstream from the point of sample introduction. When about 100 μl of sample is added to the test strip membrane on top of the area 30 that contains the colloidal gold labeled antibodies, specific binding between bacterial spores and gold labeled antibodies occurs. Simultaneously, capillary action moves the gold labeled antibodies (both spore bound and not bound) along the strip membrane 32. In the sample region 34 of the test strip 32 (downstream), specific binding of a second antibody captures bacterial spores with the attached colloidal gold labeled antibody, which gives rise to a red line in the sample region 34 due to the immobilized gold nanoparticles as shown in the bottom left of FIG. 1. In the control region 36 of the test strip 32 (further downstream), as an internal control, a polyclonal antibody binds the gold labeled antibodies that did not bind bacterial spores of interest, which also gives rise to a red line. Thus, observation of two bands, one each in the sample and control regions, indicates a positive test for the bacterial spore of interest. The observation of only one band as shown in the bottom right of FIG. 1 is a negative test result. The fundamental limitation of this method is its sensitivity; a minimum concentration of 105 spores/ml is needed before the red color from the gold nanoparticles becomes detectable; for reference, a 100 μl sample containing 10,000 anthrax spores is lethal.
Therefore what is needed is a method for improving the detection limit of lateral flow immunoassay based detection of bacterial spores, which is reported to be 105 spores/ml. This prior art detection limit prevents detection of trace quantities of bacterial spores. A trace quantity of 8000 anthrax spores, for example, is enough fill a person.
The prior art the method for determining the fraction of viable bacterial spores is based on two measurements. First, the viable bacterial spore count is measured by colony counting, and second, the total bacterial spore count is measured by direct microscopic counting. The ratio of viable to total bacterial spore count yields the fraction of spores that remain viable within a given sample. The procedure for colony counting to determine endospore concentration is comprised of the steps of (1) heat shocking the sample to kill vegetative cells while bacterial spores remain viable, (2) plating a known volume of the sample with a known dilution factor onto a growth medium, and (3) incubating the growth plates for 2 days. Finally, the resulting visible colonies are counted and reported as colony forming units (CFU's). The procedure for direct microscopic counting is comprised of the steps of (1) placing the sample on a slide with an indentation of a known volume. The glass surface of the slide is inscribed with squares of known area. (2) The bacteria in each of the several squares are counted and the average count is multiplied by an appropriate factor to yield the number of total cells per milliliter in the original suspension.
These methods suffer prohibitive difficulties with low concentration samples collected in the field. First, bacterial spores tend to attach themselves onto particulates (dust etc.) and may easily represent the bulk of the biomass in a field sample. Unfortunately, attached bacterial spores cannot be counted with either colony counting or direct microscopic counting. Second, colony-counting methods only work for cultivable bacteria, which are in the minority in field samples (<10% of microbial species form colonies). Finally, the traditional methods are lengthy (>2 days) and labor intensive. These problems have made quantification of low concentration field samples extremely difficult, and have subsequently prevented the application of these methods towards a reliable and/or real-time bacterial spore live/dead assay.
There is a need to develop a live/dead assay for bacterial spores, because there is a need to measure the fraction of bacterial spores that remain viable for samples exposed to harsh environmental conditions such as desert and arctic environments. In terms of planetary protection, which is primarily concerned with spacecraft sterilization, in order to improve sterilization procedures, one must measure the fraction of viable spores after completion of various sterilization protocols. The samples of interest contain low bacterial spore concentration and many particulates, for which the prior art methods useless.
Prior art methods for monitoring aerosolized bacterial spores includes air filtering with subsequent PCR analysis of gene segments from species of interest, and aerosol sampling with subsequent culturing and colony counting. The PCR based method is strongly dependent on impurities in the air, such as city pollution, and requires specially trained technicians to perform sample preparation prior to running the PCR reaction. The procedure for colony counting, which is comprised of (1) heat shocking the sample to kill vegetative cells while bacterial spores remain viable, (2) plating a known volume of the sample with a known dilution factor onto a growth medium, (3) incubating the growth plates for two days, also requires the active participation of a technician. This also assumes that the spore forming microbes are cultivable. It is estimated that only 10% of bacterial species are cultivable. The cost of labor, technical complexity of PCR and slow response time of colony counting have prevented the wide spread application of these methods for monitoring of bacterial spores in the air.